The purpose of this study was to compare the characteristics, physical properties, and biocompatibility of the novel tricalcium silicate-chitosan (TCS-C) sealer with AH Plus and Sure-Seal Root. The TCS-C powder was prepared by mixing tricalcium silicate with 2% water-soluble chitosan at a 5:1 ratio, followed by sufficient addition of 10 g/mL ratio of double-distilled water to form a homogeneous cement. Material characterizations (the Fourier Transform InfraRed [FTIR] and X-ray diffraction [XRD]), physical property investigations (flow and film thickness), and cytotoxicity tests in 3T3 mouse embryo fibroblast cell (MTT assay method) were performed on sealers, and the results were compared with those of the commercial products. Statistical analysis was performed on flow and film thickness. The normality of the data was tested using the Shapiro-Wilk test. Statistical analysis was performed with one-way analysis of variance (ANOVA). The level of significance was set at p < 0.05. The TCS-C showed a mean flow of 31.98 ± 0.68 mm, compared with Sure Seal Root at 26.38 ± 0.69 mm and AH Plus at 26.50 ± 0.12 mm. The TCS-C showed a mean film thickness of 60 ± 10.0 mm compared with Sure-Seal Root at 50 ± 10.0 mm and AH Plus at 40 ± 15.8 mm. The TCS-C exhibited low to no cytotoxicity in fibroblast cell at all concentrations and exposure times. Adding water-soluble chitosan may improve the physical and biologic properties of tricalcium silicate cement. The novel TCS-C sealer did not fully meet the physical properties of an endodontic sealer, but it was not cytotoxic to fibroblast cells.